Assessing GFR With Proenkephalin

Introduction In clinical practice, kidney (dys)function is monitored through creatinine-based estimations of glomerular filtration rate (eGFR: Modification of Diet in Renal Disease [MDRD], Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI]). Creatinine is recognized as a late and insensitive biomarker of glomerular filtration rate (GFR). The novel biomarker proenkephalin (PENK) may overcome these limitations, but no PENK-based equation for eGFR is currently available. Therefore, we developed and validated a PENK-based equation to assess GFR. Methods In this international multicenter study in 1354 stable and critically ill patients, GFR was measured (mGFR) through iohexol or iothalamate clearance. A generalized linear model with sigmoidal nonlinear transfer function was used for equation development in the block-randomized development set. Covariates were selected in a data-driven fashion. The novel equation was assessed for bias, precision (mean ± SD), and accuracy (eGFR percentage within ±30% of mGFR, P30) in the validation set and compared with MDRD and CKD-EPI. Results Median mGFR was 61 [44–81] ml/min per 1.73 m2. In order of importance, PENK, creatinine, and age were included, and sex or race did not improve performance. The PENK-based equation mean ± SD bias of the mGFR was 0.5 ± 15 ml/min per 1.73 m2, significantly less compared with MDRD (8 ± 17, P < 0.001) and 2009 CKD-EPI (5 ± 17, P < 0.001), not reaching statistical significance compared with 2021 CKD-EPI (1.3 ± 16, P = 0.06). The P30 accuracy of the PENK-based equation was 83%, significantly higher compared with MDRD (68%, P < 0.001) and 2009 CKD-EPI (76%, P < 0.001), similar to 2021 CKD-EPI (80%, P = 0.13). Conclusion Overall, the PENK-based equation to assess eGFR performed better than most creatinine-based equations without using sex or race.

T he measurement of creatinine to assess eGFR to represent kidney function in ambulatory and hospitalized patients is a frequently requested diagnostic test.Abnormal kidney function presages adverse clinical outcomes and is an important adjunct to the dosing of renally excreted drugs.Creatinine-based equations, such as MDRD, 1 2009 CKD-EPI, 2 and 2021 CKD-EPI, 3 are used in daily clinical practice. 4However, there are multiple shortcomings in creatinine-based calculations of eGFR, such as muscle mass and hydrational or nutritional status. 5,6In addition, both tubular reabsorption and secretion of creatinine influence the accuracy of creatinine-based eGFR equations. 4,7This may lead to not identifying those at risk of adverse health events. 8Finally, the use of race in these equations has been reevaluated, 3 and implementation of racefree equations was recently recommended. 9The true GFR can be measured using the clearance of inulin, iohexol, or iothalamate. 10However, these methods require parenteral administration, are labor intensive, and may therefore not be suited for clinical practice.
A method to estimate or measure GFR that is more accurate, precise, exempt from bias, and feasible in clinical practice remains an unmet medical need.
PENK, fully: proenkephalin A 119-159 is an endogenous protein (molecular weight w4.5 kDa), with no protein binding or cleaving known, that is purely filtered by the glomerulus and not secreted or absorbed in the tubules. 113][14][15] Many comorbidities that can occur due to or in association with acute kidney injury, such as sepsis, do not seem to influence the association of PENK with GFR. 16In a cohort of critically ill patients with septic shock, an excellent correlation was found between plasma PENK with the mGFR using iohexol plasma clearance. 17Accordingly, PENK could potentially be used to calculate eGFR which may be more accurate than creatinine-based equations.This study aimed to develop such an equation and assess its performance compared with current creatinine-based equations.

Study Design
Data were collected according to the ethical principles of the Declaration of Helsinki and approved by local medical ethical committees. 17,18Subjects included were healthy and tested before organ donation, or had a stable kidney function which was measured either on an outpatient clinic setting: patients with and without risk factors for chronic kidney disease (CKD) and after organ donation (predominately kidney donation), or were critically ill and admitted to an intensive care unit: post-cardiac surgery and patients diagnosed with having sepsis or septic shock according to the sepsis-3 criteria. 19Demographic variable "race" was collected because of the covariate in current creatinine-based equations and was self-reported.

GFR Assessment
GFR was measured using iohexol plasma clearance or iothalamate renal clearance using the gold standard method of each compound and obtained as part of clinical practice or research purposes. 10,20These compounds were measured using liquid chromatography with tandem mass spectrometry. 21,22Before (baseline) and after i.v.iohexol administration or s.c.iothalamate injection, multiple blood and urine samples were acquired from 0.75 to 6 hours post-administration. 20,23,24 Blood samples were acquired in EDTA-coated tubes after the redistribution phase, during the "slow phase" from 1.5 to 6 hours post-administration, to reconstruct the disappearance curve of iohexol. 20,23The slopeintercept method was used to determine the clearance of the iohexol, and the Bröchner-Mortensen correction was applied to adjust the redistribution phase. 23,25The calculated GFR was then corrected for body surface area. 25,26Iothalamate was injected s.c. after which iothalamate clearance was determined through iothalamate measurement in plasma and urine samples from 0.75 at 1.5 to 2 hours post-administration. 24 The calculated GFR was then corrected for body surface area. 26For comparison between equations, eGFR was calculated using the MDRD, 27 2009 CKD-EPI eGFR Cr , 28 and 2021 CKD-EPI eGFR Cr 3 equations.

Laboratory Measurements
During the GFR assessments, plasma samples were collected for PENK and creatinine concentration measurement using the sphingotest penKid sandwich immunoassay (SphingoTec GmbH, Hennigsdorf, Germany), as described previously. 21Blood was drawn in EDTA-coated tubes and was centrifuged before aliquotation of the plasma for storage at À80 C until batch analysis.Mouse monoclonal anti-PENK 152 to 159 antibodies were coated to white polystyrene microtiter plates as the capture antibody, and mouse monoclonal anti-PENK 129 to 144 antibody labeled with MACN-Acridinium-NHS ester was used as the tracer antibody.The lower detection limit was 7 pmol/ l, and the mean interassay coefficient of variation was 5.7% in the measuring range of 10.9 to 686.

Equation Validation
The newly developed equation was evaluated using a validation data set.The performance was tested in the total validation cohort and in subgroups of stable patients and critically ill patients with sepsis and those with post-cardiac surgery.The equation was assessed for bias, accuracy, and precision, through Bland-Altman analyses.For bias, the mean and predictedkidney function differences were analyzed, depicted as mGFR minus eGFR (negative bias reflects overestimation), and tested using a paired t test.Furthermore, the Lin's concordance correlation coefficient was used. 29For precision, the SD of the differences between true and eGFR was assessed.Bland-Altman analyses were conducted. 30For accuracy, the percentage of predicted kidney function between a AE30% range of the true kidney function was determined (P30) 28 and tested with the McNemar test for paired proportions.
To enable clinical decision-making, the 95% and 50% prediction intervals were calculated for which a quantile regression was used to develop a model for the prediction of mGFR at the quantiles 2.5th, 10th, 25th, 50th, 75th, 90th, and 97.5th. 31The prediction intervals were calculated as the median difference of the 2.5th and 97.5th quantiles for 95% prediction interval and median difference between the 25th and 75th quantiles for 50% prediction interval.Equation performance was compared with the widely used creatinine-based MDRD 1 and 2009 CKD-EPI equations. 28The race-free 2021 CKD-EPI eGFR Cr equation was published while this analysis was ongoing 3 and was therefore also compared with the new PENK-based equation.Furthermore, the European Kidney Function Consortium 32 and revised Lund-Malmö 33 equations were used for comparison with the PENK equation as secondary endpoints.Statistical significance was accounted to P values of <0.05.

RESULTS
The GFR was measured by gold standard methods (mGFR) in 1354 patients (Table 1 illustrating that also patients with a hyperdynamic circulation and increased renal blood flow were included in the critically ill cohorts. 34Patients in the validation set had similar baseline demographic characteristics as the development set (Table 1).
Pearson's correlation coefficient with the mGFR was 0.79 (P < 0.001) for MDRD, 0.84 (P < 0.001) for 2009 CKD-EPI, and 0.85 (P < 0.001) for 2021 CKD-EPI.Apart from PENK, available variables were tested through bootstrap iterations, using absolute and logarithmic (10) representation of continuous variables.The correlation coefficient of proenkephalin with the mGFR was 0.76, followed by creatinine at 0.75 and age at 0.17 (Table 2).Logarithmic representation of the data resulted in the most accurate equations according to SaddlePoint-Signature output.In a model with all variables, PENK was last removed using a stepwise iterative removal method, indicating that it is the most important variable (Figure 1a).Thereafter, the root mean square error was tested using a stepwise iterative introduction method; PENK narrowed the root mean square error most significantly, followed by creatinine and age (Figure 1b).Again, indicating that PENK is the most important variable for the eGFR equation building.Nominal covariates describing "race" and "age" did not further improve the performance of the equations.Regression models were applied using a linear and nonlinear form with input or output noise.The optimal model used a sigmoidal nonlinear transfer function and considered output noise.The covariates included in the equation in order of importance were as follows: PENK, creatinine, and age, all in log(10) (Figure 1).See Table 2 for the PENK-Crea equation, together with the PENK-only equation for situations when creatinine is not available, measurable, or representative, for example, in hyperbilirubinemia, neuromuscular diseases, muscle atrophy, or amputees.

Equation Validation
A new equation (Proenkephalin-Creatinine eGFR, PENK-Crea) was developed using a combined group of all stable and critically ill patients (n ¼ 811).The equation was tested for performance in the total validation set (n ¼ 543) and separately in validation subgroups of stable patients (n ¼ 456) and critically ill patients (n ¼ 87).Contour plots illustrate the impact of each parameter in the PENK-Crea equation on the calculated eGFR, compared with 2021 CKD-EPI (Supplementary Figures S2 and S3).See Supplementary Table S1 for the performance of the PENK-only, the European Kidney Function Consortium 32 and revised Lund-Malmö 33 equations.

DISCUSSION
In this study, a novel equation to assess eGFR using proenkephalin was developed and validated in a broad cohort including healthy organ donors, stable outclinic patients, patients with CKD, and critically ill patients.
The new PENK-Crea equation to calculate the eGFR performed better than widely used creatinine-based equations, both in bias and accuracy.Overall performance was similar to or better than the newly developed 2021 CKD-EPI equation.Finally, the addition of sex and race did not further improve the performance of the equation.
Multiple large observational studies have revealed that increases in PENK concentration predict and are associated with the occurrence of acute kidney injury. 13,35Given the association of renal dysfunction with unfavorable clinical outcomes, 36 this association may explain why values of PENK, when measured at hospital or intensive care unit admission, also exert predictive value for mortality. 14,37,38However, the value of PENK to reflect the actual GFR has only been described sparsely and mostly in small cohorts. 17,21,39n the present study, for the first time, PENK was correlated with gold standard GFR measurements, in a large cohort of patients, with kidney functions ranging from low to high GFR values and from stable to critically ill patients.Despite previous indications of better performance of PENK to estimate renal function, no equation to calculate eGFR based on PENK concentrations was available up to now.This equation may facilitate biomarker implementation 40 and translate PENK concentrations that the clinician may not be familiar with into a more accurate GFR estimation for physicians to work with.This also allows easier comparison to other functional biomarkers that reflect GFR, such as creatinine and cystatin C.
The analyses for covariate selection for the equation revealed that PENK concentration was most strongly correlated to mGFR and resulted for the larger part in the narrowing of the root mean square error of the equation, which underlines the more accurate To enable clinical decision making, this figure depicts the range of predicted mGFR of a patient with a given eGFR using the CKD staging cut-off points.The mGFR was predicted using a model created using quintile regression of each of the 4 equations PENK-Crea, MDRD, 2009 CKD-EPI and 2021 CKD-EPI.The Box Plots represent quantiles (minimum, first quartile, median, third quartile, and maximum) of each equation.The performance of an equation is better when the median of the predicted mGFR is closest to the given eGFR cut-off points, and when the distribution of the predicted mGFR is more narrow at that eGFR cut-off point.As an example, with an eGFR of 60 ml/min/1.73m 2 calculated with the PENK-Crea equation, 95% of the mGFR's will be in the range of 34-88.While an eGFR of 60 calculated with the MDRD equation, 95% of the mGFR's will be between the range of 37-104.mGFR or eGFR: measured or estimated glomerular filtration rate.CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; MDRD, Modification of Diet in Renal Disease.
reflection of PENK of the GFR.The addition of creatinine and age resulted in further relevant improvements.The MDRD and 2009 CKD-EPI equations also include sex and race.The use of the latter is controversial, 3,41 and therefore the new 2021 CKD-EPI equation was developed.The 2021 CKD-EPI equation without race performed better than the 2009 CKD-EPI in our cohort, whereas PENK-Crea overall performance was better or similar compared with 2021 CKD-EPI, noteworthy in patients with a GFR in the 30-60 ml/min per 1.73 m 2 .Especially in this range, dose adjusting of renally cleared drugs becomes relevant, especially in critically ill patients or hospitalized patients with CKD prone to develop acute-on-chronic kidney injury.
In critically ill patients, it is notoriously unreliable to accurately assess kidney function based on creatinine, as active tubular secretion of creatinine may conceal acute decreases in GFR.Furthermore, critically ill patients lose muscle mass within days, leading to reduced creatinine production and overestimating the eGFR.Proenkephalin avoids these issues 16 ; therefore, they may account for the observed differences between proenkephalin and creatinine-based equations.The mGFR of critically ill patients was predominantly higher compared with the mGFR of the stable patients probably because only a proportion of patients had acute kidney injury and other patients may have had an augmented renal clearance during a hyperdynamic circulation.Furthermore, a significant proportion of the stable patients were diagnosed with having CKD which was not the case for the critically ill patients.In the critically ill patients, the mGFR was measured shortly after sepsis diagnosis or shortly after cardiac surgery.Therefore, the underestimation of both the novel PENK-Crea and creatinine-based calculations of the eGFR in the critically ill patients may illustrate the acknowledged lag time of creatinine as a biomarker for GFR.Therefore, further development and validation of the equation using PENK without creatinine may be of interest for critically ill patients.
A limitation of our study is that the PENK-Crea equation has yet to be validated in larger cohorts of critically ill patients, as the number of critically ill patients in our validation cohort was relatively small.Nevertheless, a strength of this study is that the cohort used for equation development and validation included a broad range of patients, including healthy potential kidney donors, patients with established CKD, and critically ill patients.Consequently, the equation can be applied to a wide range of patients.Second, gold standard methods to determine the true mGFR were used in all patients of the cohort.Furthermore, the data-driven approach applied to develop the equation ensures an unbiased selection of covariates.However, even though this cohort consisted of multiple patient types and was block randomized in a blinded fashion to create a representative internal validation cohort, the performance of the PENK-Crea equation is still to be validated in external cohorts.
Although both exogenous compounds iohexol and iothalamate are considered the most accurate methods to measure GFR, 10,20 a cohort with only 1 gold standard method to assess the GFR would have been more optimal.Unfortunately, it is difficult to identify large cohorts with mGFR, especially outside clinical investigation or with a broad range of patients; subsequently, most equations, including CKD-EPI equations, include data on mGFR by multiple methods. 3,27,28A direct comparison between the 2 gold-standard methods is hampered by the fact that iohexol was used in critically ill patients and iothalamate clearance in stable patients in our study.Although our cohort provided subgroups large enough for covariate eligibility testing, a limitation of this study is that the subgroups differ in size.Furthermore, a comparison to equations using cystatin C, which can be accurate in specific patient groups, 42 is of interest.However, several factors that influence cystatin C production, such as corticosteroid use and inflammation, 43,44 are acknowledged, and cystatin C and its equations are not frequently used in clinical practice.Especially in critically ill patients, corticosteroids and inflammations that may influence the accuracy of cystatin C are present in many patients.Therefore, we compared PENK-Crea to the widely used creatininebased equations.Nevertheless, the absence of comparison with a cystatin C-based equation is a limitation of this study, and further testing of the PENK-Crea equation in cohorts also including cystatin C measurements is warranted.Finally, to study the possible benefit of more precise and accurate estimation of the GFR by the PENK-Crea equation in clinical practice will require pharmacokinetic studies that, for example, assess renally cleared drugs. 45Especially in critically ill patients, demonstration that a more precise calculation of eGFR will translate to a clinical outcome benefit will be challenging, although consensus guidelines [46][47][48] make it clear that a more timely and accurate estimation of GFR is a relevant unmet medical need.
In conclusion, we developed an equation to calculate eGFR using the novel biomarker for kidney function PENK, with creatinine and age.Overall, this PENK-Crea equation performs better to calculate GFR compared with most conventional or more novel creatinine-based equations.Furthermore, the frequently used covariates sex and race, of which the latter is under scrutiny, did not further improve the performance of the equation and are therefore not included in the PENK-Crea equation.These results warrant validation in external patient cohorts that will facilitate future studies into possible clinical benefits of more accurate GFR measurement.

DISCLOSURE
RB has received a travel reimbursement from SphingoTec.PP has received travel and consultancy reimbursement from SphingoTec.JCL has received grants from Allena, Arkray, Siemens, and Retrophin; others from Federation Bio, Novobiome, and Orfan-Bridgebio; and grants and others from Alnylam Pharmaceuticals, Dicerna Pharmaceuticals, OxThera, and Synlogic.ASJ presently or has in the past consulted for most of the major diagnostic companies and SphingoTec.CCW, JS, and BA are employed by SphingoTec GmbH.The proenkephalin measurements were conducted by SphingoTec, GmbH.The remaining authors declare no competing interests.

Figure 1 .
Figure 1.Equation development.(a) Figure with on the y-axis eight different covariates, with their color representing the b-coefficientas depicted in the legend.On the x-axis is the number of the covariate in the model.A higher coefficient represents a higher positive correlation of the covariate with the GFR.In a stepwise iterative removal method, covariates were first all included in the model and then removed in the sequence of importance, starting with the lowest contribution to the model's performance (African-American).The most important covariates were log(10) proenkephalin, log(10) creatinine, and age log(10).(b) RMSE (Root Mean Square Error) of the model with on the x-axis the number of covariates on the model.The covariates were added in a stepwise-enter method, in the sequence of importance, starting with log(10) proenkephalin.A lower RMSE value illustrates a more accurate predicting model.After adding covariate log (10) proenkephalin, log(10) creatinine, and log(10) age, the RMSE does not significantly improve any further.PENK, proenkephalin.

Figure 3 .
Figure 3. Scatter plots of the estimated GFR of all equations versus the measured GFR.Scatter plot of the association between the glomerular filtration rate measured by iohexol or iothalamate clearance (mGFR) and the eGFR calculated using the PENK-Crea equation, the conventional MDRD and 2009 CKD-EPI equations and the new 2021 CKD-EPI equation (eGFR) in the validation cohort.Bias is defined as the mean difference with standard deviation (in ml/min/1.73m 2 ), P30 accuracy as the percentage of estimated GFRs that is within a AE30% range of the mGFR, and correct GFR category as the percentage of patients that are correctly classified in GFR categories (all in ml/min/1.73m 2 ): "G4-5, #29 ml/min/1.73m 2 ," "G3a-b, 30-59 ml/min/1.73m 2 ," G2, "60-89 ml/min/1.73m 2 ," and "G1, $90 ml/min/1.73m 2 ."The nonlinear fit is calculated using a 2-phase association.GFR, glomerular filtration rate.

Figure 4 .
Figure 4. Distribution of the predicted mGFR at given eGFR cut-off points.To enable clinical decision making, this figure depicts the range of predicted mGFR of a patient with a given eGFR using the CKD staging cut-off points.The mGFR was predicted using a model created using quintile regression of each of the 4 equations PENK-Crea, MDRD, 2009 CKD-EPI and 2021 CKD-EPI.The Box Plots represent quantiles (minimum, first quartile, median, third quartile, and maximum) of each equation.The performance of an equation is better when the median of the predicted mGFR is closest to the given eGFR cut-off points, and when the distribution of the predicted mGFR is more narrow at that eGFR cut-off point.As an example, with an eGFR of 60 ml/min/1.73m 2 calculated with the PENK-Crea equation, 95% of the mGFR's will be in the range of 34-88.While an eGFR of 60 calculated with the MDRD equation, 95% of the mGFR's will be between the range of 37-104.mGFR or eGFR: measured or estimated glomerular filtration rate.CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; MDRD, Modification of Diet in Renal Disease.

Table 1 .
Patient baseline demographic characteristics GFR, glomerular filtration rate.Data are presented as frequency (percentage) or median [interquartile range].GFR was measured using gold standard iothalamate or iohexol clearance.Conversion factors for units: serum creatinine in mg/dl to mmol/l, Â88.42.R Beunders et al.: Calculation of Kidney Function With Proenkephalin CLINICAL RESEARCH Kidney International Reports (2023) 8, 2345-2355

Table 2 .
Covariates and the PENK-Crea equationFor the equations, creatinine was used in the mmol/l format.For use with mg/dl, multiply creatinine values with 88.42 before log(10)transformation.The PENK-only equation can be used when creatinine is not available, measurable or representative, e.g., in hyperbilirubinemia, neuromuscular diseases, muscle atrophy or in amputees, see supplement results tableS1for the performance of the PENK-only equation.